Identification and analysis of the germin-like gene family in soybean

Germin is a protein marker that was first discovered in the germination of wheat seeds [1]. Subsequently, germin and germin-like proteins (GLPs) were found in other monocotyledonous, several dicotyledonous, angiosperms, gymnospermous plants, a myxomycete (slime mould) and Physarum polycephalum[2–10]. Germin relatives have also been identified in fern spores, prokaryotes and animals [11, 12].

The germin family comprises a group of proteins belonging to a superfamily. All germins contain the germin motif that gives rise to a predicted β-barrel core involved in metal binding [13]. Most of them share biochemical attributes such as seed storage proteins, globulins and sucrose-binding, though they differ in their tissue specificities and enzyme activities [14–18]. The germin genes seemed to be involved in various important processes including development, osmotic regulation, photoperiodic oscillation, defence and apoptosis [19], and also founded to be associated with cell wall deposition [5, 7, 20, 21].

Germin has an oxalate oxidase (EC 1.2.3.4) activity [1]. There has been growing evidence that germin encoded an enzyme that degraded oxalate to CO₂ and H₂O₂ and also releases Ca⁺⁺ in some plant species. The degraded residual H₂O₂ plays different roles: a molecular signal for the induction of defence mechanisms, cross-linking of polymers in the extracellular matrix synthesis [9], and a direct antimicrobial effect, such as lignifications, to reinforce the cell walls [22–24]. The germin protein in monocotyledonous appeared to have an oxalate oxidase activity [21], but the germin-like proteins in dicotyledonous plants did not appear to have oxalate oxidase activity by 2010 [19]. For example, wheat and barley germin genes were found in the apoplast and the cytoplasm of germinating embryo cells with oxalate oxidase activity [21]. Two genes (gf-2.8 and gf-3.8) and a transcript (cDNA) of wheat germin have been sequenced [1].

Some germin genes may have functions other than oxalate oxidase activity [25]. Germin-like gene mRNAs have been found in leaves, cotyledons, stems, roots, embryos, flowers, seeds, and some were produced in response to environmental stimuli, depending on the species or the genes under consideration. Several evidences suggested that some GLPs have functions in general plant defence responses [26]. For instance, infection with pathogens, feeding of insects or application of chemicals such as salicylic acid, hydrogen peroxide (H₂O₂) or ethylene [27–32] could increase the expression of GLPs. In wheat and barley, transcription of at least one germin gene was induced upon a fungal infection [33]. Endogenous factors also controlled the expression of some germin genes since transcription of wheat germin gf-2.8 gene is stimulated by auxins [20]. Transient overexpression and transient silencing of certain barley GLP genes resulted in enhanced resistance to the powdery mildew fungus [17]. The promoter variant of rice oxalate oxidase genes played a role in resistance to Magnaporthe oryzae[34]. For some subfamilies, transient and stable expression showed a superoxide dismutase activity (EC1.15.1.1) of the encoded protein [31]. Silencing of a Nicotiana attenuata GLP increased the performance of an herbivore [30]. mRNA levels of mustard (Brassica napus) and a closely related Arabidopsis germin gene fluctuated during the circadian cycle [5, 8].

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a serious fungal disease of soybean. Since oxalic acid is a major pathogenic factor of SSR, transgenic soybean capable of degrading oxalic acid may be resistant to the pathogen [35]. To date, the wheat gf-2.8 gene has been studied on resistance to the oxalate-secreting pathogen S. sclerotiorum. Results showed transgenic soybean with the wheat germin gene greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum, indicating that the germin gene products conferred resistant to Sclerotinia stem rot [35].

The GLPs with mostly unknown function in plant genomes [26] had been classified into subfamilies [36, 37]. For example, the true germin subfamily, such as wheat and barley germins, included proteins with oxalate oxidase activity. In contrast, both GLP subfamilies 1 and 2 contained examples of proteins with superoxide dismutase (SOD) activity. Subfamily 3 included the phosphodiesterase (EC3.1) activity described above, and more subdivisions had been proposed recently [38]. A key feature of the GLP-related subfamilies, including the germins [37], was the conservation of a motif derived from that of the cupin superfamily [36]. In barley, five GLP subfamilies have been described and named HvGER1 to HvGER5. In Physcomitrella patens germin-like proteins, two novel clades have been found, named bryophyte-subfamilies 1 and 2 [38].

In contrast to the advanced knowledge of the structure, cell biology and expression features of barley and wheat germins and GLPs, less was known about soybean germin and germin-like genes by 2010. In the present work, nearly 50,000 sequenced and annotated ESTs of soybean were analyzed to find GmGER-like sequences by amino acid sequence similarity, and GmGER-like sequences were elongated by RACE. Their locations were determined and compared to disease resistance QTL, and both cluster and phylogenetic analyses of the germin-like proteins were performed to describe the variations in the soybean gene family. The abiotic factors (auxin-IAA, salt, light treatments) was tested on GmGER genes to evaluate the complex biological processes related to soybean development.

Germin genes form a large family and their functions are still under studying. In this study, we identified 21 germin genes of soybean. The analyses of gene domain revealed that the unknown GLP gene family and their functions might be determined according to the presence of germin domain. Interestingly, the predicted model almost perfectly conformed to the determined structure of individual domain of the seed storage globulins, canavalin and phaseolin that showed a significant sequence similarity with germins [39, 40]. These proteins contain two or three similar domains that might have evolved following gene duplication events from a common ancestral gene or domain [40, 41].

To date, no germin gene was identified in soybean. Thus, the 21 GmGER genes found in the present work were thought as the novel, species-specific proteins of soybean. Comparative analyses revealed that these evolutionary related germin genes shared very similar exon structures, suggested the close phylogenetic relationships among the soybean germin genes.

The common structural features of germin-like proteins included: conserved structural elements, secretory transit peptides, protein glycosylation sites and regions of conserved sequence similarity [36]. According to the review of Carter and Thornburg [36], the most important conserved structural feature among the GLPs is a conserved amino acid sequence termed the germin box. The consensus pattern is: GxxxxHxHPxAxEh, where x is any amino acid and h is a hydrophobic amino acid. Mature germins and GLPs all contain approximately 200 amino acids in length and the germin box occurs near the middle for all proteins in this family. In soybean, the conserved region B was found in all the 21 germin genes. Conserved region A enriched in hydrophobic amino acids always followed this peptide (GxxxxHxHPxAxEh) [Figure 2]. Another conserved region A was localized in the amino terminal part of the mature proteins, and strongly conserved in all the germins but not in the spherulins. It is likely that the heptapeptide sequence [L/V]QDFCV[A/G], found in all germin-like proteins examined, was of importance in the biochemical functions of these proteins. A third putative conserved region C underlined in Figure 2 was [V/M][F/K]P[Q/K/I]G[L][V/I/L]HFQ[K/L/Q/I]N[V/N/I]G. Two His residues and a Glu residue in Motif A, together with a His residue in Motif C, act as ligands for the binding of a manganese ion at the active site of the archetypal germin, cupin [42]. In our result, motif A was HTHPRATEILTVLEGTLYVGF with 21 amino acids, motif B was KVLNKGDVFVFPEGLIHFQFN with 21 amino acids, and motif C was [N/S]SQNPGIVFVPLTLFG with 16 amino acids. The 21 GmGER genes all have the germin activity, e xhibiting diverse expression patterns during soybean development, a regular photoperiodical reaction in darkness, response to abiotic stress (such as auxin and salt), as we have proved in this paper.

One question that remained to be answered was whether all germin or germin-like genes of soybean carried out the same functions and were regulated in the same way like in other species. Multi-sequence alignments of GLPs in barley and Arabidopsis, as well in other plant species showed some overlaps of multigene family structure, which would be helpful in functional annotation and the study of the evolutionary relationships among the genes [31]. Based on phylogenetic analysis, we concluded that the GmGER genes did not share a common expression pattern. Germin genes were functionally diverse but structurally related [43]. Large differences existed among several members of germin gene family. It was clear that soybean GmGER genes were homologous to the dicotyledon proteins in the phylogenetic tree (Figure 1). Sequence analyses revealed that GmGER 8, 9 and 10 were distinct from the other GmGER genes. The GmGER 8 and 9 were located on the same chromosome 7, suggesting that they might be divergence from a common ancestor. Likewise, the GmGER 2, 3, 6, 11, 12, 13, 14 and 20 were closely related to each other. It was possible that they were recently descended from a common ancestor gene that evolved into different forms.

Germin and the GLP gene family could be divided into two distinct groups. The members in one group (germins) had relatively homogeneous sequences [44], meanwhile, the members in another group (GLPs) were much more numerous and showed high sequence divergence [45]. GLPs could be further divided into three subgroups based upon sequence conservation [37]. In this paper, the GmGER genes were also divided into three subgroups based upon sequence conservation. Systematic investigation of soybean germin gene family would be useful for defining origins of the germin and germin-like gene family. Although the functional significance of all these elements remained to be tested, a variety of regulatory mechanisms acting on the transcript abundance were found. The germin genes in rice have been confirmed to be expressed in all types of tissues and could be induced by biotic or abiotic stresses. Many of the stress-induced germin genes were physically co-localized with quantitative trait loci (QTL) for disease resistance, and the emerged evidence suggested that microRNAs might regulate their transcript abundances [46, 47]. Oxalate oxidase could confer to enhance resistance to Sclerotinia blight in peanut [41]. In transformed sunflower, the expression of oxalate oxidase resulted in the induction of plant defense proteins [48]. In tissue sections derived from pea nodules, PsGER1 was shown to be the first known germin-like protein with superoxide dismutase activity [49].

A significant promotion of the expression of GmGER mRNA was achieved by quantitative real-time RT-PCR under the supplement of IAA in medium. As IAA is involved mainly in the regulation of cell elongation and stimulating cell division [50], this result suggested that GmGER genes might mediate the stimulating effect of auxin on cell division. It should be noticed that, although the GmGER gene responded to IAA, the affinity was unknown, which made it difficult to be certain that the GmGER gene was involved in auxin signaling, especially without additional physiologic evidence. Therefore, it should be investigated furthermore. Meanwhile, the assay of salt stress indicated that the transgenic seedlings of tobacco with GmGER 9 gene showed improved salt tolerance compared to the WT plants (Figure 7A, 7B, 7C, 7D, 7E and 7F), confirming that the GmGER 9 gene had a positive response to the salt condition. Salt stress could cause oxidative damage in plant, such as protein oxidation and lipid peroxidation [51]. Therefore, GmGER 9 gene remained a potential to improve the salt tolerance in transgenic plants.

Different families of genes have been reported to be associated with plant photoperiod including germin genes [19]. A significant difference was observed in the response to light/dark cycles by various genes and different species. The level of mRNA of Sinapis alba L. undergoes circadian oscillation during light/dark cycles with a maximum about 12 h after the light was turned on. This peak of mRNA accumulation occurred in light treatment [52]. By contrast, the level of GmGER mRNA reached its maximum about 12 h after light was turned off in this study. The peak of mRNA accumulation occurred during the dark period in darkness treatment and changed sharply. In the light treatment the mRNA had almost no expression. In the LD treatment, the mRNA level had mild change less than in SD treatment. The value of mRNA was a little higher in SD treatment than in LD treatment. These findings indicated that the transcription of the GmGER genes greatly depended on the duration of darkness.

In summary, 21 germin-like protein genes of soybean were identified and analyzed in this study. The results revealed that this novel family of germin-like proteins might represent an important mechanism in soybean to modulate diverse physiological and molecular processes. These findings provided the groundwork to assist functional studies of this novel GmGER family, and an opportunity to discover the roles of the germin family proteins, to underlie regulatory mechanisms during plant development and the responses to adverse environmental stimuli and to answer why several different genes were required to carry out these functions. Future studies using molecular, genetic, biochemical, physiological, and other approaches could provide insights into understanding the functions and elucidating the molecular mechanisms of soybean germin genes in plant defense responses and development.